Ion Exchange Chromatography Question

[neutron1]

A mix of Cytochrome C, Myoglobin, and Alkaline phosphatase are placed into a gel chromatography column filled with DEAE, a positively charged stationary phase. 2 different Tris buffers at pH of 8 are used to elute different fractions. The first buffer has a NaCl concentration of 0.15 molar, while buffer 2 has a NaCl concentration of 0.5 molar. Of the three collected fractions, which protein for the original mix would end up in which fraction?

Basically, which of the 3 proteins elutes out in what order because of the use of increasing salt concentration for the buffer. Can somebody please provide assistance! Biochemistry is not my strong point at all. Thanks in advance 🙂

---

Members don't see this ad.

 

[aldol16]

You can't answer this question without knowing something more about each protein. When we run ion exchange columns, we have to have some inkling of the pI of each protein. If you don't have information, it's like taking a shot in the dark, hoping you hit something.

 

[Ad2b]

You can't answer this question without knowing something more about each protein.

The 3 proteins are listed. Cyto-C, myoglobin, alkaline phosphatase; with PIs of 10, 7, 8.0 respectively.

I'm no expert on ion exchange but I think the order would be CytoC, AP, then myo... given the PIs and the buffer of pH 8.0

Looking forward to someone else chiming in and explaining it better/correcting me.

 

[IWillMakeIt1]

When pH>pI, you are positively charged, this is an anionic-exchange chromatography since you have a positive charge resin (DEAE) so the negative would be attracted to DEAE and the positively charged protein will be eluted first. so myoglobin, AP, then cyto-C given the PIs above is correct.

 

[neutron1]

The 3 proteins are listed. Cyto-C, myoglobin, alkaline phosphatase; with PIs of 10, 7, 8.0 respectively.

I'm no expert on ion exchange but I think the order would be CytoC, AP, then myo... given the PIs and the buffer of pH 8.0

Looking forward to someone else chiming in and explaining it better/correcting me.

When pH>pI, you are positively charged, this is an anionic-exchange chromatography since you have a positive charge resin (DEAE) so the negative would be attracted to DEAE and the positively charged protein will be eluted first. so myoglobin, AP, then cyto-C given the PIs above is correct.

In my research,

Cytochrome C pi= 10
Myoglobin = 7
Alkaline Phosphatase = 4.5

Assuming this order, the elution rate from no buffer, buffer 1, and buffer 2, should go from cyto-c --> Myoglobin ---> Alkaline phosphatase, correct?

 

[Danny L]

The 3 proteins are listed. Cyto-C, myoglobin, alkaline phosphatase; with PIs of 10, 7, 8.0 respectively.

I'm no expert on ion exchange but I think the order would be CytoC, AP, then myo... given the PIs and the buffer of pH 8.0

Looking forward to someone else chiming in and explaining it better/correcting me.

Do they expect us to know those PIs off the top of our head?...

Or are they given?

 

[Ad2b]

Do they expect us to know those PIs off the top of our head?... Or are they given?

Not for general molecules but should know roughly how PI would be impacted by phobic/phillic, acidic/basic, & Histidine of the amino acids. I am guessing the OP's question came from some test prep company.

The question posed by OP would be too straightforward on real MCAT.

Cytochrome C pi= 10
Myoglobin = 7
Alkaline Phosphatase = 4.5

alkaline indicates basic which is greater than 7.0... 4.5 would be a pretty acidic PI

 

[aldol16]

The 3 proteins are listed. Cyto-C, myoglobin, alkaline phosphatase; with PIs of 10, 7, 8.0 respectively.

I'm no expert on ion exchange but I think the order would be CytoC, AP, then myo... given the PIs and the buffer of pH 8.0

Looking forward to someone else chiming in and explaining it better/correcting me.

In my research,

Cytochrome C pi= 10
Myoglobin = 7
Alkaline Phosphatase = 4.5

Assuming this order, the elution rate from no buffer, buffer 1, and buffer 2, should go from cyto-c --> Myoglobin ---> Alkaline phosphatase, correct?

You are not expected to know the pIs of each protein off the top of your head even if they are listed. Unrealistic question if you are and also unanswerable unless you're in the field, which is too much to expect of pre-meds.

Anion exchange column will bind negatively-charged particles as they come through. The salt gradient you use for elution is to compete with the column for binding with the sample. The more tightly bound the molecule, the higher the salt concentration you need to effectively unbind the molecule. So of the proteins listed and OP's research, alkaline phosphatase and myoglobin will both be negatively charged at pH 8 whereas cyt C will be positively charged. Thus, cyt C will come out first because the column won't really bind to it well. Then the elution order will be based on pI. The lower the pI here, the higher the salt gradient needed because the more strongly it will bind the column. So Myo would come out next, followed by alkaline phosphatase.

However, in this case, we would generally use a pH gradient, which would give us more control over where our desired protein would elute, since we know the pIs. So you would start at pH 8 and then gradually make it more acidic so that Myo becomes protonated first and falls off and then alkaline phosphatase below pH 3 or so.

 

[aldol16]

alkaline indicates basic which is greater than 7.0... 4.5 would be a pretty acidic PI

Don't get caught up in enzyme names, as they are often non-intuitively named. Alkaline phosphatase indicates that it works best under basic conditions whereas pI gives protonation state of the enzyme itself, which is not necessarily related to enzymatic activity.

 

[aldol16]

When pH>pI, you are positively charged, this is an anionic-exchange chromatography since you have a positive charge resin (DEAE) so the negative would be attracted to DEAE and the positively charged protein will be eluted first. so myoglobin, AP, then cyto-C given the PIs above is correct.

Be careful. pI is like pKa. If pH > pI, you would be negatively charged, just as with pH > pKa. So if something has a pKa of 4 and the pH is 8, then that thing is going to act like an acid and be deprotonated (i.e. negatively charged). So the positively-charged protein is actually cyt-C.

 

[IWillMakeIt1]

Be careful. pI is like pKa. If pH > pI, you would be negatively charged, just as with pH > pKa. So if something has a pKa of 4 and the pH is 8, then that thing is going to act like an acid and be deprotonated (i.e. negatively charged). So the positively-charged protein is actually cyt-C.

yeah I messed up there 😀